Our goal is exploit the power of single molecule observation to elucidate the mechanism by which specific sequences within mRNA modulate the rate of translation by E. coli ribosomes, through use of an approach coupling Total Internal Reflection Fluorescence Microscopy (TIRFM) with Fluorescence Resonance Energy Transfer (FRET). In our approach, fluorescent groups are introduced in the ribosome that, by FRET interaction with fluorescently-labeled tRNAs, allow initial aminoacyl-tRNA binding to the ribosomal A-site, tRNA translocation to the P-site, and release of discharged tRNA from the E-site to be monitored on single ribosomes in real time. This approach will provide a detailed, continuous kinetic profile of mRNA translation during continuous elongation, providing unique insights into the regulation of translation rates in protein synthesis that are important for cell function. We will determine kinetic profiles for expression of i) short model mRNAs containing known pausing elements regulating translation; ii) complete mRNAs coding for full proteins; and iii) designed mutations of such mRNAs that permit rigorous evaluation of the effects of pausing elements, singly or in groups, in the context of full protein synthesis. Such determinations will allow new understanding of the roles such pauses play in biologically important processes and providing suggestions for optimizing cell-free protein synthesis systems. Our specific aims are to: 1. Determine translation profiles for model mRNAs. We will determine translation kinetic profiles for model mRNAs incorporating known pausing elements (rare codons, downstream mRNA 2o structure, upstream nascent peptides) either one at a time or in tandem. The information obtained will quantify effects of such elements on translation and elucidate the mechanism of the intrinsic ribosomal helicase. 2. Determine translation rates for full-length mRNAs. We will determine translation kinetic profiles for full length mRNAs and specifically designed mutants of such mRNAs in order to determine how surrounding context influences the effect of a given pausing element or group of pausing elements on translation rate, beginning with the mRNAs coding for E. coli dihydrofolate reductase (DHFR) and chloramphenicol acetyltransferase (CATIII). 3. Optimize the reagents employed in the TIRFM-FRET approach. The principal improvements over currently available reagents will be directed toward i. reducing background from fluorescently-labeled tRNA by derivatizing EF-Tu with a fluorescence quencher; ii. synthesizing a larger variety of fluorescent tRNAs; and iii. labeling ribosomes with quantum dots for increased stability toward photobleaching. 4. Optimize the apparatus and methods needed for the TIRFM-FRET approach. Several improvements to the apparatus, software and procedures will be accomplished to enable collection of long kinetic sequences from the onset of elongation, with high fidelity and minimum perturbation by photobleaching. The image processing software used to quantify single molecule FRET pairs and their efficiencies will be improved, optimized and statistically validated. PUBLIC HEALTH RELEVANCE: Three major consequences will flow from our work. First, we will be able to examine the effects of known translational pausing elements in the context of complete protein chain expression, and to determine if new elements and synergistic effects can be identified. Second, we will be able to systematically explore the role translational pausing plays in the integration of protein synthesis and cellular function, focusing on such issues as the tradeoff between speed and accuracy in translation at functionally crucial residues, and the possible coupling of translational pausing and co-translational protein folding. Third, we will be able to provide important information with respect to optimizing cell-free protein translation systems.